Yeast cells expressing TAR DNA-binding protein 43 and uses therefor

ABSTRACT

Disclosed are yeast cells expressing TAR DNA-binding protein 43 (TDP-43) and methods of screening yeast cells to identify compounds that prevent or suppress TDP-43-induced toxicity, compounds that inhibit the formation or maintenance of cytoplasmic inclusions of TDP-43, genetic suppressors or enhancers of TDP-43-induced toxicity, and genetic suppressors or enhancers of the formation or maintenance of cytoplasmic inclusions of TDP-43. Compounds identified by such screens can be used to treat or prevent TDP-43 proteinopathies such as frontotemporal lobar degeneration or amyotrophic lateral sclerosis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase filing under 35 U.S.C. 371 ofinternational application number PCT/US2009/034117, filed Feb. 13, 2009,which claims priority from U.S. Provisional Application No. 61/029,168,filed Feb. 15, 2008. The entire content of each of these priorapplications is incorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under grant number 2P50NS038372-0681. The Government has certain rights in the invention.

TECHNICAL FIELD

The invention relates to protein chemistry and cellular and molecularbiology

BACKGROUND

TDP-43 (TAR DNA-Binding Protein 43) was identified as a protein thatbinds to human immunodeficiency virus type 1 TAR DNA sequence motifs andrepresses transcription of viral RNA (Ou, S., J Virol., 69(6):3584-96,1995). TDP-43 was also identified as a splicing regulator that binds tothe (UG)m-repeated polymorphic region near the 3′ splice site of CFTRexon 9 and down-regulates its recognition by the splicing machinery(Buratti, E. and Baralle F E, J Biol Chem., 276(39):36337-43, 2001 andreferences therein). Notably, exon 9 skipping produces a nonfunctionalCFTR protein and is associated with some forms of cystic fibrosis. Itwas also observed that TDP-43 recognizes (UG) repeats in other contexts.

Hyperphosphorylated, ubiquitinated, C-terminal fragments of TDP-43 havebeen recovered from the central nervous system (e.g., hippocampus,neocortex, and spinal cord) from patients with frontotemporal lobardegeneration (FTLD) with ubiquitin-positive inclusions and amyotrophiclateral sclerosis (ALS) and is believed to be the hallmark pathologicalfeature of FTLD and ALS (Neumann et al. Science, 314(5796):130-3. 2006).The presence or amount of abnormal aggregation of phosphorylated andubiquitinated TDP-43 is believed to define a novel class ofneurodegenerative diseases referred to as “TDP-43 proteinopathies”(Cairns et al. (2007) The American Journal of Pathology 171(1):227).

SUMMARY

The invention is based, at least in part, on the discovery thatexpression of a human TDP-43 protein in yeast cells is toxic and resultsin the formation of TDP-43-containing insoluble cytoplasmic inclusions.This discovery permits the carrying out of screens usingTDP-43-expressing yeast cells to identify compounds or genetic factorsthat modulate TDP-43-induced toxicity or TDP-43-induced inclusionformation. Compounds identified by such screens can be used for thetreatment or prevention of TDP-43 proteinopathies such as frontotemporallobar degeneration or amyotrophic lateral sclerosis.

Described herein is a yeast cell containing an expression constructcontaining a promoter operably linked to a nucleic acid encoding apolypeptide containing a TAR DNA-binding protein 43 (TDP-43) protein,wherein expression of the nucleic acid and production of the polypeptideresults in a decrease in growth or viability of the cell. In someembodiments, expression of the nucleic acid and production of thepolypeptide renders the yeast cell non-viable.

The expression construct can optionally be integrated in the genome ofthe yeast cell. For example, the expression construct can be anintegrative plasmid such as pRS303, pRS304, pRS305, or pRS306. A yeastcell can have one or more (e.g., two, three, or four) integrated copiesof an expression construct.

The promoter can be an inducible promoter such as GAL1-10, GAL1, GALL,GALS, GPD, ADH, TEF, CYC1, MRP7, MET25, TET, VP16, or VP16-ER.Alternatively, the promoter can be a constitutively active promoter.

In some embodiments, the TDP-43 protein is human TDP-43. The polypeptidecan optionally contain a full length TDP-43 protein (e.g., SEQ ID NO:1).The polypeptide can be a fusion protein containing a detectable protein(e.g., a fluorescent protein, an enzyme, or an epitope). Exemplaryfluorescent proteins include red fluorescent protein, green fluorescentprotein, blue fluorescent protein, yellow fluorescent protein, and cyanfluorescent protein.

In some embodiments, the yeast is Saccharomyces cerevisiae,Saccharomyces uvae, Saccharomyces kluyveri, Schizosaccharomyces pombe,Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Pichiamethanolica, Pichia kluyveri, Yarrowia lipolytica, Candida sp., Candidautilis, Candida cacaoi, Geotrichum sp., or Geotrichum fermentans.

In some embodiments, at least one gene that encodes a protein involvedin drug efflux or cell permeability is disrupted in the yeast cell. Forexample, one or more of the genes PDR1, PDR3, PDR5, SNQ2, or ERG6 can bedisrupted in the yeast cell.

Also disclosed is a method of inducing toxicity in a yeast cell by:providing a yeast cell described herein; and inducing a level ofexpression of the nucleic acid in the yeast cell that is toxic to theyeast cell.

Also disclosed is a method of identifying a compound that prevents orsuppresses TDP-43-induced toxicity by: culturing a yeast cell describedherein in the presence of a candidate agent and under conditions thatallow for expression of the nucleic acid at a level that, in the absenceof the candidate agent, is sufficient to induce toxicity in the cell;measuring cell growth or viability in the presence of the candidateagent; and comparing cell growth or viability measured in the presenceof the candidate agent to cell growth or viability in the absence of thecandidate agent, wherein if cell growth or viability is increased in thepresence of the candidate agent as compared to in the absence of thecandidate agent, then the candidate agent is identified as a compoundthat prevents or suppresses TDP-43-induced toxicity.

Also disclosed is a method of identifying a compound that inhibits theformation or maintenance of cytoplasmic inclusions of TDP-43 by:providing a yeast cell containing an expression construct containing anucleic acid encoding a protein containing TDP-43; culturing the yeastcell in the presence of a candidate agent and under conditions thatallow for expression of the nucleic acid at a level that, in the absenceof the candidate agent, is sufficient to result in the formation ormaintenance of cytoplasmic inclusions of the protein; measuring theformation or maintenance of cytoplasmic inclusions of the protein in thepresence of the candidate agent; and comparing the formation ormaintenance of cytoplasmic inclusions of the protein measured in thepresence of the candidate agent to the formation or maintenance ofcytoplasmic inclusions of the protein in the absence of the candidateagent, wherein if formation or maintenance of cytoplasmic inclusions ofthe protein is decreased in the presence of the candidate agent ascompared to in the absence of the candidate agent, then the candidateagent is identified as a compound that inhibits the formation ormaintenance of cytoplasmic inclusions of TDP-43.

Also disclosed is a method of identifying a genetic suppressor orenhancer of TDP-43-induced toxicity by: providing a yeast cell describedherein, wherein the yeast cell has been genetically modified tooverexpress a gene; culturing the yeast cell under conditions that allowfor expression of the protein at a level that, in the absence ofoverexpression of the gene, is sufficient to induce toxicity in theyeast cell; measuring cell growth or viability in the presence ofoverexpression of the gene; and comparing cell growth or viabilitymeasured in the presence of overexpression of the gene to cell growth orviability in the absence of overexpression of the gene, wherein (i) ifcell growth or viability is increased in the presence of overexpressionof the gene as compared to in the absence overexpression of the gene,then the gene is identified as a genetic suppressor of TDP-43-inducedtoxicity, and (ii) if cell growth or viability is decreased in thepresence of overexpression of the gene as compared to in the absenceoverexpression of the gene, then the gene is identified as a geneticenhancer of TDP-43-induced toxicity.

Also disclosed is a method of identifying a genetic suppressor orenhancer of TDP-43-induced toxicity by: providing a yeast cell describedherein, wherein an endogenous gene of the yeast cell has been disrupted;culturing the yeast cell under conditions that allow for expression ofthe protein at a level that, in the absence of disruption of theendogenous gene, is sufficient to induce toxicity in the yeast cell;measuring cell growth or viability in the presence of disruption of theendogenous gene; and comparing cell growth or viability measured in thepresence of disruption of the endogenous gene to cell growth orviability in the absence of disruption of the endogenous gene, wherein(i) if cell growth or viability is increased in the presence ofdisruption of the endogenous gene as compared to in the absencedisruption of the endogenous gene, then the gene is identified as agenetic enhancer of TDP-43-induced toxicity, and (ii) if cell growth orviability is decreased in the presence of disruption of the endogenousgene as compared to in the absence disruption of the endogenous gene,then the gene is identified as a genetic suppressor of TDP-43-inducedtoxicity.

Also disclosed is a method of treating a TDP-43 proteinopathy byadministering to a subject (e.g., a human) having, or at risk ofdeveloping, a TDP-43 proteinopathy a pharmaceutical compositioncontaining a therapeutically effective amount of a compound identifiedby a method described herein.

In some embodiments, the TDP-43 proteinopathy is a frontotemporaldementia such as frontotemporal lobar degeneration. For example, thefrontotemporal dementia can be frontotemporal lobar degeneration withtau-positive inclusions or frontotemporal lobar degeneration withubiquitin-positive, tau- and alpha-synuclein negative inclusions.Frontotemporal lobar degeneration with tau-positive inclusions includefrontotemporal lobar degeneration with Pick bodies, corticobasaldegeneration, progressive supranuclear palsy, and neurofibrillarytangle-only dementia.

In some embodiments, the TDP-43 proteinopathy is amyotrophic lateralsclerosis.

Also disclosed is a method of reducing the amount, or preventing theoccurrence, of aggregate TDP-43 in a cell (e.g., a human cell) bycontacting a cell containing aggregate TDP-43 with a compound identifiedby a method described herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentapplication, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of yeast cells expressing GFP alone (A) or GFPfused to full length human TDP-43 (B). Induction of GFP-TDP-43expression resulted in the formation of multiple cytoplasmic inclusionsin the yeast cells.

FIG. 2 is a photograph of yeast cells transformed with either an emptyvector or a galactose-inducible TDP-43 expression plasmid. The cellswere spotted on glucose or galactose and growth was assessed after twodays. The transformants grew equally well on the control glucose plates(TDP-43 “off”), whereas TDP-43 expression profoundly inhibited growth(TDP-43 “on”).

DETAILED DESCRIPTION

The TDP-43-expressing yeast cells described herein can be used toidentify compounds or genetic factors that modulate TDP-43-inducedtoxicity and/or TDP-43-induced inclusion formation. Compounds identifiedby such screens can be used for the treatment or prevention ofneurodegenerative diseases characterized by abnormal aggregation ofTDP-43. For example, compounds that inhibit or reverse aggregation ofTDP-43 would be expected to be of use as therapeutic agents inneurodegenerative diseases in which TDP-43 aggregation plays a role.

TDP-43 Proteins and Nucleic Acids

Described herein are compositions and methods for identifying candidatecompounds that prevent or suppress TDP-43-induced toxicity, candidatecompounds that inhibit the formation or maintenance of cytoplasmicinclusions of TDP-43, genetic suppressors or enhancers of TDP-43-inducedtoxicity, and genetic suppressors or enhancers of the formation ormaintenance of cytoplasmic inclusions of TDP-43.

A TDP-43 protein used in the compositions and methods described hereincontains the sequence of a naturally occurring TDP-43 polypeptide or abiologically active fragment or variant thereof. A “biologically active”fragment or variant of a naturally occurring TDP-43 polypeptide retainsthe ability to induce a decrease cell growth or viability and/or induceformation of cytoplasmic inclusions in a yeast cell in which thefragment or variant is expressed. A biologically active fragment orvariant of a TDP-43 polypeptide can contain one or more additions,substitutions, and/or deletions relative to the sequence of a naturallyoccurring TDP-43 polypeptide.

Human TDP-43 is 414 amino acids in length and has the following aminoacid sequence (GenBank™ Reference NP_031401):

(SEQ ID NO: 1) MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVEGILHAPDAGWGNLVYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPWKTTEQDLKEYFSTFGEVLMVQVKKDLKTGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWCDCKLPNSKQSQDEPLRSRKVFVGRCTEDMTEDELREFFSQYGDVMDVFIPKPFRAFAFVTFADDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNPGGFGNQGGFGNSRGGGAGLGNNQGSNMGGGMNFGAFSINPAMMAAAQAALQSSWGMMGMLASQQNQSGPSGNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGSGFNGGFGSSMDSKSSGWGM.

C-terminal fragments of human TDP-43 have been recovered from thecentral nervous system of patients with frontotemporal lobardegeneration with ubiquitin-positive inclusions and amyotrophic lateralsclerosis (Neumann et al. Science, 314(5796):130-3. 2006). The diseaseassociated C-terminal fragments of TDP-43 correspond to approximatelyamino acid residues 252-414 of SEQ ID NO:1.

In some embodiments, a variant TDP-43 polypeptide (i) contains one ormore amino acid substitutions, and (ii) is at least 70%, 80%, 85%, 90%,95%, 98% or 99% identical to SEQ ID NO:1 (or 70%, 80%, 85%, 90%, 95%,98% or 99% identical to amino acids 252-414 of SEQ ID NO:1). A variantTDP-43 polypeptide differing in sequence from SEQ ID NO:1 (or differingin sequence from amino acids 252-414 of SEQ ID NO:1) may include one ormore amino acid substitutions (conservative or non-conservative), one ormore deletions, and/or one or more insertions. In an exemplaryembodiment, a biologically active TDP-43 fragment contains amino acidresidues 252-414 of SEQ ID NO:1.

Nucleic acids encoding variant TDP-43 polypeptides can be used toidentify those variants that exhibit enhanced toxicity in yeast cellsrelative to that exhibited by the human TDP-43 protein of SEQ ID NO:1.Such enhanced toxicity variants are of use in the screening and othermethods described herein.

A fusion protein containing a TDP-43 protein may be used. For example, aTDP-43 protein may be fused with a second domain. The second domain ofthe fusion protein can optionally be an immunoglobulin element, adimerizing domain, a targeting domain, a stabilizing domain, or apurification domain. Alternatively, a TDP-43 protein can be fused with aheterologous molecule such as a detection protein. Exemplary detectionproteins include: a fluorescent protein such as green fluorescentprotein (GFP), cyan fluorescent protein (CFP) or yellow fluorescentprotein (YFP); an enzyme such as β-galactosidase or alkaline phosphatase(AP); and an epitope such as glutathione-S-transferase (GST) orhemagglutinin (HA). To illustrate, a TDP-43 protein can be fused to GFPat the N- or C-terminus or other parts of the TDP-43 protein. Thesefusion proteins provide methods for rapid and easy detection andidentification of the TDP-43 protein in the recombinant yeast cell.

Human TDP-43 is encoded by the following nucleotide sequence (GenBank™Reference NM_007375):

(SEQ ID NO: 2) GGTGGGCGGGGGGAGGAGGCGGCCCTAGCGCCATTTTGTGGGAGCGAAGCGGTGGCTGGGCTGCGCTTGGGTCCGTCGCTGCTTCGGTGTCCCTGTCGGGCTTCCCAGCAGCGGCCTAGCGGGAAAAGTAAAAGATGTCTGAATATATTCGGGTAACCGAAGATGAGAACGATGAGCCCATTGAAATACCATCGGAAGACGATGGGACGGTGCTGCTCTCCACGGTTACAGCCCAGTTTCCAGGGGCGTGTGGGCTTCGCTACAGGAATCCAGTGTCTCAGTGTATGAGAGGTGTCCGGCTGGTAGAAGGAATTCTGCATGCCCCAGATGCTGGCTGGGGAAATCTGGTGTATGTTGTCAACTATCCAAAAGATAACAAAAGAAAAATGGATGAGACAGATGCTTCATCAGCAGTGAAAGTGAAAAGAGCAGTCCAGAAAACATCCGATTTAATAGTGTTGGGTCTCCCATGGAAAACAACCGAACAGGACCTGAAAGAGTATTTTAGTACCTTTGGAGAAGTTCTTATGGTGCAGGTCAAGAAAGATCTTAAGACTGGTCATTCAAAGGGGTTTGGCTTTGTTCGTTTTACGGAATATGAAACACAAGTGAAAGTAATGTCACAGCGACATATGATAGATGGACGATGGTGTGACTGCAAACTTCCTAATTCTAAGCAAAGCCAAGATGAGCCTTTGAGAAGCAGAAAAGTGTTTGTGGGGCGCTGTACAGAGGACATGACTGAGGATGAGCTGCGGGAGTTCTTCTCTCAGTACGGGGATGTGATGGATGTCTTCATCCCCAAGCCATTCAGGGCCTTTGCCTTTGTTACATTTGCAGATGATCAGATTGCGCAGTCTCTTTGTGGAGAGGACTTGATCATTAAAGGAATCAGCGTTCATATATCCAATGCCGAACCTAAGCACAATAGCAATAGACAGTTAGAAAGAAGTGGAAGATTTGGTGGTAATCCAGGTGGCTTTGGGAATCAGGGTGGATTTGGTAATAGCAGAGGGGGTGGAGCTGGTTTGGGAAACAATCAAGGTAGTAATATGGGTGGTGGGATGAACTTTGGTGCGTTCAGCATTAATCCAGCCATGATGGCTGCCGCCCAGGCAGCACTACAGAGCAGTTGGGGTATGATGGGCATGTTAGCCAGCCAGCAGAACCAGTCAGGCCCATCGGGTAATAACCAAAACCAAGGCAACATGCAGAGGGAGCCAAACCAGGCCTTCGGTTCTGGAAATAACTCTTATAGTGGCTCTAATTCTGGTGCAGCAATTGGTTGGGGATCAGCATCCAATGCAGGGTCGGGCAGTGGTTTTAATGGAGGCTTTGGCTCAAGCATGGATTCTAAGTCTTCTGGCTGGGGAATGTAGACAGTGGGGTTGTGGTTGGTTGGTATAGAATGGTGGGAATTCAAATTTTTCTAAACTCATGGTAAGTATATTGTAAAATACATATGTACTAAGAATTTTCAAAATTGGTTTGTTCAGTGTGGAGTATATTCAGCAGTATTTTTGACATTTTTCTTTAGAAAAAGGAAGAGCTAAAGGAATTTTATAAGTTTTGTTACATGAAAGGTTGAAATATTGAGTGGTTGAAAGTGAACTGCTGTTTGCCTGATTGGTAAACCAACACACTACAATTGATATCAAAAGGTTTCTCCTGTAATATTTTATCCCTGGACTTGTCAAGTGAATTCTTTGCATGTTCAAAACGGAAACCATTGATTAGAACTACATTCTTTACCCCTTGTTTTAATTTGAACCCCACCATATGGATTTTTTTCCTTAAGAAAATCTCCTTTTAGGAGATCATGGTGTCACAGTGTTTGGTTCTTTTGTTTTGTTTTTTAACACTTGTCTCCCCTCATACACAAAAGTACAATATGAAGCCTTCATTTAATCTCTGCAGTTCATCTCATTTCAAATGTTTATGGAAGAAGCACTTCATTGAAAGTAGTGCTGTAAATATTCTGCCATAGGAATACTGTCTACATGCTTTCTCATTCAAGAATTCGTCATCACGCATCACAGGCCGCGTCTTTGACGGTGGGTGTCCCATTTTTATCCGCTACTCTTTATTTCATGGAGTCGTATCAACGCTATGAACGCAAGGCTGTGATATGGAACCAGAAGGCTGTCTGAACTTTTGAAACCTTGTGTGGGATTGATGGTGGTGCCGAGGCATGAAAGGCTAGTATGAGCGAGAAAAGGAGAGAGCGCGTGCAGAGACTTGGTGGTGCATAATGGATATTTTTTAACTTGGCGAGATGTGTCTCTCAATCCTGTGGCTTTGGTGAGAGAGTGTGCAGAGAGCAATGATAGCAAATAATGTACGAATGTTTTTTGCATTCAAAGGACATCCACATCTGTTGGAAGACTTTTAAGTGAGTTTTTGTTCTTAGATAACCCACATTAGATGAATGTGTTAAGTGAAATGATACTTGTACTCCCCCTACCCCTTTGTCAACTGCTGTGAATGCTGTATGGTGTGTGTTCTCTTCTGTTACTGATATGTAAGTGTGGCAATGTGAACTGAAGCTGATGGGCTGAGAACATGGACTGAGCTTGTGGTGTGCTTTGCAGGAGGACTTGAAGCAGAGTTCACCAGTGAGCTCAGGTGTCTCAAAGAAGGGTGGAAGTTCTAATGTCTGTTAGCTACCCATAAGAATGCTGTTTGCTGCAGTTCTGTGTCCTGTGCTTGGATGCTTTTTATAAGAGTTGTCATTGTTGGAAATTCTTAAATAAAACTGATTTAAATAATATGTGTCTTTGTTTTGCAGCCCTGAATGCAAAGAATTCATAGCAGTTAATTCCCCTTTTTTGACCCTTTTGAGATGGAACTTTCATAAAGTTTCTTGGCAGTAGTTTATTTTGCTTCAAATAAACTTATTTGAAAAGTTGTCTCAAGTCAAATGGATTCATCACCTGTCATGCATTGACACCTGATACCCAGACTTAATTGGTATTTGTTCTTGCATTGGCCAAAGTGAAAATTTTTTTTTTTCTTTTGAAATCTAGTTTTGAATAAGTCTGGGTGACCGCACCTAAAATGGTAAGCAGTACCCTCCGGCTTTTTCTTAGTGCCTCTGTGCATTTGGGTGATGTTCTATTTACATGGCCTGTGTAAATCTCCATTGGGAAGTCATGCCTTCTAAAAAGATTCTTATTTGGGGGAGTGGGCAAAATGTTGATTATTTTCTAATGCTTTGTAGCAAAGCATATCAATTGAAAAGGGAATATCAGCACCTTCCTAGTTTGGGATTTGAAAAGTGGAATTAATTGCAGTAGGGATAAAGTAGAAGAAACCACAAATTATCTTGTGCCTGAAATCCATTAAGAGGCCTGATAGCTTTAAGAATTAGGGTGGGTTGTCTGTCTGGAAGTGTTAAGTGGAATGGGCTTTGTCCTCCAGGAGGTGGGGGAATGTGGTAACATTGAATACAGTTGAATAAAATCGCTTACAAAACTCACACTCTCACAATGCATTGTTAAGTATGTAAAAGCAATAACATTGATTCTCTGTTGTACTTTTTTGTAACTAATTCTGTGAGAGTTGAGCTCATTTTCTAGTTGGAAGAATGTGATATTTGTTGTGTTGGTAGTTTACCTAATGCCCTTACCTAATTAGATTATGATAAATAGGTTTGTCATTTTGCAAGTTACATAAACATTTATCAATGAAGTCATCCTTTAGACTTGTAATCGCCACATTGTTTCATTATTCAGTTTCCTCTGTAAAGGGATCTTGAGTTGTTTTAATTTTTTTTTTCTGCATCTGAATCTGCATGATTTCCAAACCCTGTACCATCTGAATTTTGCATTTTAGCACTTGCACTATTACTCAGCAGCAGTAACATGGTAACACTTAAAATGGTACTCGGGGACCTCCAAAGACTAAACTGACAAGCCTTCAAGGAGCCCAGGGGTAAGTTAACTTGTCAACGGCATGGTTTAATCCCTTCTTTACACTTGTGTAAATTTCAGTTACTGGTCATAGAAGGCTTTCAATGTTGAGTGGCCTTTTATTAACATGTTTATGGTACTGCATAGATACGGGTATTTATTTTACCCTAAGAAGATTTTGAAGTTTAAAAGTACTTAAACTATTTGGCAAAGATTTGTTTTTAAAAATCTATTTGGTCAATCTAAATGCATTCATTCTAAAAAATTTTTTGAACCAGATAAATAAAATTTTTTTTTGACACCACAAAAAAAA AAAAAAAAAAAA.The coding region extends from nucleotides 135-1379 of SEQ ID NO: 2.

Also described herein are methods of preparing and transferring nucleicacids encoding a TDP-43 protein into a cell so that the cell expressesthe TDP-43 protein. The term “TDP-43 nucleic acid” encompasses a nucleicacid containing a sequence as represented in SEQ ID NO:2 as well as anucleic acid encoding any of the biologically active fragments orvariants of TDP-43 described herein. Exemplary TDP-43 nucleic acidsinclude those encoding full length human TDP-43 and C-terminal fragmentsthereof.

The term “nucleic acid” generally refers to at least one molecule orstrand of DNA, RNA or a derivative or mimic thereof, containing at leastone nucleobase, for example, a naturally occurring purine or pyrimidinebase found in DNA or RNA. Generally, the term “nucleic acid” refers toat least one single-stranded molecule, but in specific embodiments willalso encompass at least one additional strand that is partially,substantially or fully complementary to the at least one single-strandedmolecule. Thus, a nucleic acid may encompass at least onedouble-stranded molecule or at least one triple-stranded molecule thatcomprises one or more complementary strand(s) or “complement(s)” of aparticular sequence comprising a strand of the molecule.

Yeast Cells

Yeast strains that can be used in the compositions and methods describedherein include, but are not limited to, Saccharomyces cerevisiae,Saccharomyces uvae, Saccharomyces kluyveri, Schizosaccharomyces pombe,Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Pichiamethanolica, Pichia kluyveri, Yarrowia lipolytica, Candida sp., Candidautilis, Candida cacaoi, Geotrichum sp., and Geotrichum fermentans.Although much of the discussion herein relates to Saccharomycescerevisiae which ectopically expresses an abnormally processed protein,this is merely for illustrative purposes. Other yeast strains can besubstituted for S. cerevisiae.

Certain aspects of the disclosure relate to screening methods foridentifying candidate therapeutic agents (e.g., pharmaceutical,chemical, or genetic agents). The methods described herein canoptionally be carried out in yeast strains bearing mutations in the ERG6gene, the PDR1 gene, the PDR3 gene, the PDR5 gene, the SNQ2 gene, and/orany other gene which affects membrane efflux pumps and/or increasespermeability for drugs.

A nucleic acid encoding a TDP-43 protein may be transfected into a yeastcell using nucleic acid vectors that include, but are not limited to,plasmids, linear nucleic acid molecules, artificial chromosomes, andepisomal vectors.

Three well known systems used for recombinant plasmid expression andreplication in yeast cells include integrative plasmids, low-copy-numberARS-CEN plasmids, and high-copy-number 2μ plasmids. See Sikorski,“Extrachromosomal cloning vectors of Saccharomyces cerevisiae,” inPlasmid, A Practical Approach, Ed. K. G. Hardy, IRL Press, 1993; andYeast Cloning Vectors and Genes, Current Protocols in Molecular Biology,Section II, Unit 13.4, Eds., Ausubel et al., 1994.

An example of the integrative plasmids is YIp, which is maintained atone copy per haploid genome, and is inherited in Mendelian fashion. Sucha plasmid, containing a gene of interest, a bacterial origin ofreplication and a selectable gene (typically an antibiotic-resistancemarker), is produced in bacteria. The purified vector is linearizedwithin the selectable gene and used to transform competent yeast cells.

An example of the low-copy-number ARS-CEN plasmids is YCp, whichcontains the autonomous replicating sequence (ARS1) and a centromericsequence (CEN4). These plasmids are usually present at 1-2 copies percell. Removal of the CEN sequence yields a YRp plasmid, which istypically present in 100-200 copies per cell. However, this plasmid isboth mitotically and meiotically unstable.

An example of the high-copy-number 2μ plasmids is YEp, which contains asequence approximately 1 kb in length (named the 2μ sequence). The 2μsequence acts as a yeast replicon giving rise to higher plasmid copynumber. However, these plasmids are unstable and require selection formaintenance. Copy number is increased by having on the plasmid aselection gene operatively linked to a crippled promoter.

A wide variety of plasmids can be used in the compositions and methodsdescribed herein. In one embodiment, the plasmid is an integrativeplasmid (e.g., pRS303, pRS304, pRS305 or pRS306 or other integrativeplasmids). In further embodiments, the plasmid is an episomal plasmid(e.g., p426GPD, p416GPD, p426TEF, p423GPD, p425GPD, p424GPD or p426GAL).

Regardless of the type of plasmid used, yeast cells are typicallytransformed by chemical methods (e.g., as described by Rose et al.,1990, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.). The cells are typically treated with lithiumacetate to achieve transformation efficiencies of approximately 10⁴colony-forming units (transformed cells)/μg of DNA. Yeast performhomologous recombination such that the cut, selectable marker recombineswith the mutated (usually a point mutation or a small deletion) hostgene to restore function. Transformed cells are then isolated onselective media. Of course, any suitable means of introducing nucleicacids into yeast cells can be used.

The yeast vectors (plasmids) described herein typically contain a yeastorigin of replication, an antibiotic resistance gene, a bacterial originof replication (for propagation in bacterial cells), multiple cloningsites, and a yeast nutritional gene for maintenance in yeast cells. Thenutritional gene (or “auxotrophic marker”) is most often one of thefollowing: 1) TRP1 (Phosphoribosylanthranilate isomerase); 2) URA3(Orotidine-5′-phosphate decarboxylase); 3) LEU2 (3-Isopropylmalatedehydrogenase); 4) HIS3 (Imidazoleglycerolphosphate dehydratase or IGPdehydratase); or 5) LYS2 (α-aminoadipate-semialdehyde dehydrogenase).

The yeast vectors (plasmids) described herein may also contain promotersequences. A “promoter” is a control sequence that is a region of anucleic acid sequence at which initiation and rate of transcription arecontrolled. It may contain genetic elements at which regulatory proteinsand molecules may bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription a nucleic acid sequence.The phrases “operatively linked” and “operatively positioned” mean thata promoter is in a correct functional location and/or orientation inrelation to a nucleic acid sequence to control transcriptionalinitiation and/or expression of that sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Alternatively, a promoter may be arecombinant or heterologous promoter, which refers to a promoter that isnot normally associated with a nucleic acid sequence in its naturalenvironment. Such promoters may include promoters of other genes andpromoters not “naturally occurring.” The promoters employed may beeither constitutive or inducible.

For example, various yeast-specific promoters (elements) may be employedto regulate the expression of a RNA in yeast cells. Examples ofinducible yeast promoters include GAL1-10, GAL1, GALL, GALS, TET, VP16and VP16-ER. Examples of repressible yeast promoters include Met25.Examples of constitutive yeast promoters include glyceraldehyde3-phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter(ADH), translation-elongation factor-1-alpha promoter (TEF), cytochromec-oxidase promoter (CYC1), and MRP7. Autonomously replicating expressionvectors of yeast containing promoters inducible by glucocorticoidhormones have also been described (Picard et al., 1990), including theglucocorticoid responsive element (GRE). These and other examples aredescribed in Mumber et al., 1995; Ronicke et al., 1997; Gao, 2000, allincorporated herein by reference. Yet other yeast vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. and Grantet al., 1987.

Screening Assays

Certain aspects of the present disclosure provide methods of screeningfor a candidate drug (agent or compound) or a genetic factor thatmodulates TDP-43-induced toxicity or TDP-43-induced inclusion formation.Various types of candidate drugs may be screened by the methodsdescribed herein, including nucleic acids, polypeptides, small moleculecompounds, and peptidomimetics. In some cases, genetic agents can bescreened by contacting the yeast cell with a nucleic acid constructcoding for a gene. For example, one may screen cDNA libraries expressinga variety of genes, to identify genes that modulate TDP-43-inducedtoxicity or TDP-43-induced inclusion formation.

For example, the identified drugs may modulate TDP-43-induced toxicityor TDP-43-induced inclusion formation. Accordingly, irrespective of theexact mechanism of action, drugs identified by the screening methodsdescribed herein are expected to provide therapeutic benefit to TDP-43proteinopathies.

In certain embodiments, screening methods described herein use yeastcells that are engineered to express a TDP-43 protein. For chemicalscreens, suitable mutations of yeast strains designed to affect membraneefflux pumps and increase permeability for drugs can be used. Forexample, a yeast strain bearing mutations in the ERG6 gene, the PDR1gene, the PDR3 gene, and/or the PDR5 gene is contemplated of use. Forexample, a yeast strain bearing mutations in membrane efflux pumps(erg6, pdr1, pdr3, and/or pdr5) has been successfully used in manyscreens to identify growth regulators (Jensen-Pergakes K L, et al.,1998. Antimicrob Agents Chemother 42:1160-7).

In certain embodiments, candidate drugs can be screened from largelibraries of synthetic or natural compounds. One example is an FDAapproved library of compounds that can be used by humans. In addition,compound libraries are commercially available from a number of companiesincluding but not limited to Maybridge Chemical Co. (Trevillet,Cornwall, UK), Comgenex (Princeton, N.J.), Microsource (New Milford,Conn.), Aldrich (Milwaukee, Wis.), AKos Consulting and Solutions GmbH(Basel, Switzerland), Ambinter (Paris, France), Asinex (Moscow, Russia),Aurora (Graz, Austria), BioFocus DPI, Switzerland, Bionet (Camelford,UK), ChemBridge, (San Diego, Calif.), ChemDiv, (San Diego, Calif.),Chemical Block Lt, (Moscow, Russia), ChemStar (Moscow, Russia),Exclusive Chemistry, Ltd (Obninsk, Russia), Enamine (Kiev, Ukraine),Evotec (Hamburg, Germany), Indofine (Hillsborough, N.J.), Interbioscreen(Moscow, Russia), Interchim (Montlucon, France), Life Chemicals, Inc.(Orange, Conn.), Microchemistry Ltd. (Moscow, Russia), Otava, (Toronto,ON), PharmEx Ltd. (Moscow, Russia), Princeton Biomolecular (MonmouthJunction, N.J.), Scientific Exchange (Center Ossipee, N.H.), Specs(Delft, Netherlands), TimTec (Newark, Del.), Toronto Research Corp.(North York ON), UkrOrgSynthesis (Kiev, Ukraine), Vitas-M, (Moscow,Russia), Zelinsky Institute, (Moscow, Russia), and Bicoll (Shanghai,China). Combinatorial libraries are available and can be prepared.Libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are commercially available or can be readilyprepared by methods well known in the art. It is proposed that compoundsisolated from natural sources, such as animals, bacteria, fungi, plantsources, including leaves and bark, and marine samples may be assayed ascandidates for the presence of potentially useful pharmaceutical agents.It will be understood that the pharmaceutical agents to be screenedcould also be derived or synthesized from chemical compositions orman-made compounds. Several commercial libraries can be used in thescreens.

Another embodiment relates to genetic screens. For example, genomiclibraries and disruption libraries can be screened to find extragenicsuppressors or enhancers of TDP-43-induced toxicity or TDP-43-inducedinclusion formation. Because the yeast genome is small, 10,000transformants of each type should be sufficient for good coverage.

One embodiment contemplates screening assays using fluorescent resonanceenergy transfer (FRET). FRET occurs when a donor fluorophore is in closeproximity (10-60 A) to an acceptor fluorophore, and when the emissionwavelength of the first overlaps the excitation wavelength of the second(Kenworthy A K et al., 2001. Methods. 24:289-96). FRET should occur whencyan fluorescent protein (CFP) and yellow fluorescent protein (YFP)fusion proteins are actually part of the same complex.

For example, a TDP-43 protein can be fused to CFP and to YFPrespectively, and integrated in the yeast genome under the regulation ofa GAL1-10 promoter. Cells are grown in galactose to induce expression.Upon induction, cells produce the fusion proteins, which aggregate andbring the CFP and YFP close together. Because proteins in the aggregatesare tightly packed, the distance between the CFP and YFP is less thanthe critical value of 100 A that is necessary for FRET to occur. In thiscase, the energy released by the emission of CFP will excite the YFP,which in turn will emit at its characteristic wavelength. FRET basedscreening can be used to identify candidate compounds including, drugs,genes or other factors that can disrupt the interaction of CFP and YFPby maintaining the proteins in a state that does not allow aggregationto occur.

One embodiment contemplates screening assays using fluorescenceactivated cell sorting (FACS) analysis. FACS is a technique well knownin the art, and provides the means of scanning individual cells for thepresence of fluorescently labeled/tagged moiety. The method is unique inits ability to provide a rapid, reliable, quantitative, andmultiparameter analysis on either living or fixed cells. For example, aTDP-43 protein can be suitably labeled, and provide a useful tool forthe analysis and quantitation of protein aggregation as a result ofother genetic or growth conditions of individual yeast cells asdescribed above.

In particular embodiments, methods of the present disclosure relate todetermining TDP-43-induced toxicity. One of the strongest aspects ofyeast is the possibility of performing high throughput screens that mayidentify genes, peptides and other compounds with the potential toameliorate toxicity. A large number of compounds can be screened under avariety of growth conditions and in a variety of genetic backgrounds.The toxicity screen has the advantage of not only selecting forcompounds that interact with TDP-43, but also upstream or downstreamtargets that are not themselves cytotoxic and that are not yetidentified.

For example, the Bioscreen-C system (Labsystem) permits the growth of upto 200 cell cultures at the same time, under different conditions.Growth rates are monitored optically, recorded automatically, and storedas digital files for further manipulations. Growth can be monitored inthe presence of genetic libraries, chemicals, drugs, etc. to identifythose that give a selective growth advantage or disadvantage. Mutantsand chemicals from a variety of sources will be tested.

Certain embodiments provide methods of further testing those potentialdrugs that have been identified in the yeast system, in other modelsystems. The model systems include, but are not limited to, worms,flies, mammalian cells, and in vivo animal models.

Compounds

Compounds to be screened or identified using any of the methodsdescribed herein can include various chemical classes, though typicallysmall organic molecules having a molecular weight in the range of 50 to2,500 daltons. These compounds can comprise functional groups necessaryfor structural interaction with proteins (e.g., hydrogen bonding), andtypically include at least an amine, carbonyl, hydroxyl, or carboxylgroup, and preferably at least two of the functional chemical groups.These compounds often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures (e.g., purinecore) substituted with one or more of the above functional groups.

In alternative embodiments, compounds can also include biomoleculesincluding, but not limited to, peptides, polypeptides, peptidomimetics(e.g., peptoids), amino acids, amino acid analogs, saccharides, fattyacids, steroids, purines, pyrimidines, derivatives or structuralanalogues thereof, polynucleotides, nucleic acid aptamers, andpolynucleotide analogs.

Compounds can be identified from a number of potential sources,including: chemical libraries, natural product libraries, andcombinatorial libraries comprised of random peptides, oligonucleotides,or organic molecules. Chemical libraries consist of diverse chemicalstructures, some of which are analogs of known compounds or analogs orcompounds that have been identified as “hits” or “leads” in other drugdiscovery screens, while others are derived from natural products, andstill others arise from non-directed synthetic organic chemistry.Natural product libraries re collections of microorganisms, animals,plants, or marine organisms which are used to create mixtures forscreening by: (1) fermentation and extraction of broths from soil, plantor marine microorganisms, or (2) extraction of plants or marineorganisms. Natural product libraries include polypeptides, non-ribosomalpeptides, and variants (non-naturally occurring) thereof. For a review,see Science 282:63-68 (1998). Combinatorial libraries are composed orlarge numbers of peptides, oligonucleotides, or organic compounds as amixture. These libraries are relatively easy to prepare by traditionalautomated synthesis methods, PCR, cloning, or proprietary syntheticmethods. Of particular interest are non-peptide combinatorial libraries.Still other libraries of interest include peptide, protein,peptidomimetic, multiparallel synthetic collection, recombinatorial, andpolypeptide libraries. For a review of combinatorial chemistry andlibraries created therefrom, see Myers, Curr. Opin. Biotechnol.8:701-707 (1997).

Identification of test compounds through the use of the variouslibraries herein permits subsequent modification of the test compound“hit” or “lead” to optimize the capacity of the “hit” or “lead” toprevent or suppress TDP-43-induced toxicity and/or TDP-43-inducedinclusion formation.

The compounds identified above can be synthesized by any chemical orbiological method. The compounds identified above can also be pure, ormay be in a heterologous composition (e.g., a pharmaceuticalcomposition), and can be prepared in an assay-, physiologic-, orpharmaceutically-acceptable diluent or carrier (see below).

Pharmaceutical Compositions

A compound that is found to prevent or suppress TDP-43-induced toxicityor the formation of TDP-43 aggregates (e.g., one that prevents orsuppresses the formation, deposition, accumulation, or persistence ofubiquitinated, hyperphosphorylated forms of TDP-43 protein and/orC-terminal fragments thereof) in a cell can be formulated as apharmaceutical composition, e.g., for administration to a subject totreat a TDP-43 proteinopathy such as frontotemporal lobar degeneration(FTLD), amyotrophic lateral sclerosis (ALS), certain types of motorneuron disease (MND), or any other TDP-43 proteinopathy.

A pharmaceutical composition typically includes a pharmaceuticallyacceptable carrier. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Thecomposition can include a pharmaceutically acceptable salt, e.g., anacid addition salt or a base addition salt (see e.g., Berge et al., J.Pharm. Sci. 66:1-19, 1977).

The compound can be formulated according to standard methods.Pharmaceutical formulation is a well-established art, and is furtherdescribed, e.g., in Gennaro (ed.), Remington: The Science and Practiceof Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000) (ISBN:0683306472); Ansel et al., Pharmaceutical Dosage Forms and Drug DeliverySystems, 7th Ed., Lippincott Williams & Wilkins Publishers (1999) (ISBN:0683305727); and Kibbe (ed.), Handbook of Pharmaceutical ExcipientsAmerican Pharmaceutical Association, 3rd ed. (2000) (ISBN: 091733096X).

In one embodiment, a compound that prevents or suppresses TDP-43-inducedtoxicity and/or TDP-43 aggregate formation in a cell can be formulatedwith excipient materials, such as sodium chloride, sodium dibasicphosphate heptahydrate, sodium monobasic phosphate, and a stabilizer. Itcan be provided, for example, in a buffered solution at a suitableconcentration and can be stored at 2-8° C.

The pharmaceutical compositions may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, capsules, pills, powders, liposomes andsuppositories. The preferred form can depend on the intended mode ofadministration and therapeutic application. Typically compositions forthe agents described herein are in the form of injectable or infusiblesolutions.

Such compositions can be administered by a parenteral mode (e.g.,intravenous, subcutaneous, intraperitoneal, or intramuscular injection).The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and include, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural, intracerebral, intracranial,intracarotid and intrasternal injection and infusion.

The composition can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable for stablestorage at high concentration. Sterile injectable solutions can beprepared by incorporating an agent described herein in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating acompound into a sterile vehicle that contains a basic dispersion mediumand the required other ingredients from those enumerated above. In thecase of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying that yields a powder of a compound plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

In certain embodiments, the compound can be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known. See, e.g.,Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson,ed., Marcel Dekker, Inc., New York, 1978.

A compound identified as one that prevents or suppresses TDP-43-inducedtoxicity and/or TDP-43 aggregate formation in a cell can be modified,e.g., with a moiety that improves its stabilization and/or retention incirculation, e.g., in blood, serum, or other tissues, e.g., by at least1.5, 2, 5, 10, or 50 fold. The modified compound can be evaluated toassess whether it can reach treatment sites of interest (e.g., locationsof aggregate TDP-43) such as can occur in a cell in a subject with aTDP-43 proteinopathy such as ALS (e.g., by using a labeled form of thecompound).

For example, the compound can be associated with a polymer, e.g., asubstantially non-antigenic polymer, such as a polyalkylene oxide or apolyethylene oxide. Suitable polymers will vary substantially by weight.Polymers having molecular number average weights ranging from about 200to about 35,000 Daltons (or about 1,000 to about 15,000, and 2,000 toabout 12,500) can be used. For example, a compound can be conjugated toa water soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g.,polyvinylalcohol or polyvinylpyrrolidone. A non-limiting list of suchpolymers include polyalkylene oxide homopolymers such as polyethyleneglycol (PEG) or polypropylene glycols, polyoxyethylenated polyols,copolymers thereof and block copolymers thereof, provided that the watersolubility of the block copolymers is maintained. Additional usefulpolymers include polyoxyalkylenes such as polyoxyethylene,polyoxypropylene, and block copolymers of polyoxyethylene andpolyoxypropylene (Pluronics); polymethacrylates; carbomers; and branchedor unbranched polysaccharides.

When the compound is used in combination with a second agent (e.g., anyadditional therapies for a TDP-43 proteinopathy such as a decongestantor Rilutek®), the two agents can be formulated separately or together.For example, the respective pharmaceutical compositions can be mixed,e.g., just prior to administration, and administered together or can beadministered separately, e.g., at the same or different times aselaborated below.

Methods for Treating a TDP-43 Proteinopathy

Disclosed herein are methods to treat or prevent a TDP-43 proteinopathy.In practicing the methods, effective amounts of the compounds orcompositions described herein are administered. Such amounts aresufficient to achieve a therapeutically effective concentration of thecompound or active component of the composition in vivo. The methods canbe in vitro or in vivo methods.

A TDP-43 proteinopathy treated or whose symptoms are ameliorated by thecompounds and compositions described is a disease associated with theformation, deposition, accumulation, or persistence of forms (e.g.,ubiquitinated, hyperphosphorylated forms) of TDP-43 protein and/orC-terminal fragments thereof. (See, e.g., Cairns et al. (2007) TheAmerican Journal of Pathology 171(1):227). In certain embodiments, suchdiseases include, e.g., frontotemporal dementia (FTD) (e.g.,frontotemporal lobar degeneration (FTLD)), amyotrophic lateral sclerosis(ALS), and certain types of motor neuron disease (MND). An FTLD can be,e.g.: (i) FTLD with tau-positive inclusions such as FTLD with Pickbodies, corticobasal degeneration (CBD), progressive supranuclear palsy(PSP0, or neurofibrillary tangle-only dementia; or (ii) FTLD withubiquitin-positive, tau- and alpha-synuclein negative inclusions (UBI).(See, e.g., Kwong et al. (2007) Acta Neuropathol. 114:63-70). A TDP-43proteinopathy can be a sporadic or familial (inherited) disorder (e.g.,a sporadic or familial form of FTLD or ALS).

As used herein, a subject “suspected of having a TDP-43 proteinopathy”is one who exhibits one or more symptoms of a TDP-43 proteinopathy.Symptoms of a TDP-43 proteinopathy can include one or more of a varietyof behavioral abnormalities such as, e.g., apathy or an unwillingness totalk; change in personality and mood (e.g., depression); lack ofinhibition or lack of social tact; obsessive or repetitive behavior(e.g., compulsively shaving or collecting items); unusual verbal,physical, or sexual behavior; dramatic overeating; poor hygiene; and ageneral lack of awareness that any behavioral changes have occurred.Subjects affected by a TDP-43 proteinopathy can also manifest speech ormemory abnormalities such as difficulty speaking (or finding a correctword), reading, and/or writing. Symptoms also include neurologicalabnormalities such as, e.g., twitching, cramping, muscle stiffness,muscle weakness, impaired balance, abnormal muscle postures, poorcoordination, difficulty swallowing, and/or slurred speech. Any of thesymptoms described above can range in severity, but generally increasein severity as a TDP-43 proteinopathy progresses.

A subject “at risk of developing” a TDP-43 proteinopathy is one with afamily history of a TDP-43 proteinopathy and/or a genetic predispositionfor developing a TDP-43 proteinopathy. For example, mutations inprogranulin are associated with approximately 5-10% of all cases of FTDand mutations in superoxide dismutase 1 (SOD1) are associated withapproximately 20% of familial ALS (see, e.g., Bruni et al. (2007)Neurology 69:140-147 and Battistini et al. (2005) J. Neurol.252(7):782-8). Additional genetic mutations associated with familial andsporadic forms of ALS are described in Pasinelli et al. (2006) Nat. Rev.Neurosci. 7(9):710-23 and Gros-Louis et al. (2006) Biochim. Biophys.Acta. 1762(11-12):956-72. In some embodiments, a subject having a familyhistory for developing a TDP-43 proteinopathy has one or more of themutations identified as being associated with the particularproteinopathy (e.g., FTD or ALS). In other cases, a subject having afamily history for developing a TDP-43 proteinopathy has only a medicalrecord of the family history but has not been identified as having aparticular genetic mutation associated with the proteinopathy (e.g., theindividual has a family history of ALS but has a wild type SOD1 geneand/or lacks an SOD1 mutation that has been associated with ALS).

Methods for determining whether a subject has a TDP-43 proteinopathyinclude any of a number of qualitative and quantitative tests. Forexample, a medical practitioner (e.g., a doctor or nurse) can review themedical history of a subject and/or determine the number or type ofsymptoms that a particular subject presents. In some instances, amedical practitioner will perform one or more tests to rule out otherconditions that have similar phenotypes. For example, a practitioner canperform any of an electromyography (EMG), a nerve conduction velocity(NCV) test, a magnetic resonance imaging (MRI) test, or a blood or urinetest.

A compound useful for treating, preventing, or ameliorating one or moresymptoms of a TDP-43 proteinopathy can be administered to a subject,e.g., a human subject, by a variety of methods. For many applications,the route of administration is one of: intravenous injection or infusion(IV), subcutaneous injection (SC), intraperitoneally (IP), orintramuscular injection. In some cases, administration can be directlyinto the CNS, e.g., intrathecal, intracerebroventricular (ICV),intracerebral, or intracranial. The compound can be administered as afixed dose, or in a mg/kg dose. In other instances, administration canbe oral (e.g., administered by inhalation), transdermal (topical),transmucosal, or rectal.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

The powders and tablets contain from 1% to 95% (w/w) of the activecompound. In certain embodiments, the active compound ranges from 5% to70% (w/w). Suitable carriers are magnesium carbonate, magnesiumstearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin,tragacanth, methylcellulose, sodium carboxymethylcellulose, a lowmelting wax, cocoa butter, and the like. The term “preparation” isintended to include the formulation of the active compound withencapsulating material as a carrier providing a capsule in which theactive component with or without other carriers, is surrounded by acarrier, which is thus in association with it. Similarly, cachets andlozenges are included. Tablets, powders, capsules, pills, cachets, andlozenges can be used as solid dosage forms suitable for oraladministration.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizers, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,and other well-known suspending agents.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

Where the compound is a polypeptide or otherwise particularly antigenic,the dose can also be chosen to reduce or avoid production of antibodiesagainst the compound. The route and/or mode of administration of theagent can also be tailored for the individual case.

Dosage regimens are adjusted to provide the desired response, e.g., atherapeutic response or, when administered in combination with anotheragent, a combinatorial therapeutic effect. The dosage regimen can, forexample, be capable of treating, preventing, or ameliorating one or moresymptoms of a TDP-43 proteinopathy (or prevent or suppress theformation, deposition, accumulation, or persistence of ubiquitinated,phosphorylated (or hyperphosphorylated) forms of TDP-43 protein and/orC-terminal fragments thereof in one or more affected cells in a subjecthaving a TDP-43 proteinopathy). The dose of a compound can be,optionally, formulated separately or together with an appropriate doseof a second therapeutic agent can be used to provide a subject with theagent. Suitable dosages and/or dose ranges for the compound include anamount sufficient to treat, prevent, or ameliorate one or more symptomsof a TDP-43 proteinopathy (or prevent or suppress the formation,deposition, accumulation, or persistence of ubiquitinated,hyperphosphorylated forms of TDP-43 protein and/or C-terminal fragmentsthereof in one or more affected cells in a subject having a TDP-43proteinopathy). Such dosages can include, e.g., about 0.001 μg/kg to10,000 μg/kg body weight of the subject, per dose. In another example,the dosage can be about 1 μg/kg to 100 μg/kg body weight of the subject,per dose. In another example, the dosage can be about 1 μg/kg to 30μg/kg body weight of the subject, per dose, e.g., from 3 μg/kg to 10μg/kg body weight of the subject, per dose.

A dose of a compound required to treat, prevent, or ameliorate one ormore symptoms of a TDP-43 proteinopathy (or prevent or suppress theformation, deposition, accumulation, or persistence of ubiquitinated,hyperphosphorylated forms of TDP-43 protein and/or C-terminal fragmentsthereof in one or more affected cells in a subject having a TDP-43proteinopathy) can depend on a variety of factors including, forexample, the age, sex, and weight of a subject to be treated. Otherfactors affecting the dose administered to the subject include, e.g.,the type or severity of the subject's TDP-43 proteinopathy. For example,a patient with advanced ALS can require a administration of a differentdosage of a compound than a patient with a milder form of ALS. Otherfactors can include, e.g., other disorders concurrently or previouslyaffecting the subject, the general health of the subject, the geneticdisposition of the subject, diet, time of administration, rate ofexcretion, drug combination, and any other additional therapeutics thatare administered to the subject. It should also be understood that aspecific dosage and treatment regimen for any particular subject willdepend upon the judgment of the treating medical practitioner. Theamount of any active ingredients will also depend upon the particulardescribed compound and the presence or absence and the nature of theadditional therapeutic agents in a composition containing the compound.

Dosage unit form or “fixed dose” as used herein refers to physicallydiscrete units suited as unitary dosages for the subjects to be treated;each unit contains a predetermined quantity of active compoundcalculated to produce the desired therapeutic effect (e.g., treating,preventing, or ameliorating one or more symptoms of a TDP-43proteinopathy (or preventing or suppressing the formation, deposition,accumulation, or persistence of ubiquitinated, hyperphosphorylated formsof TDP-43 protein and/or C-terminal fragments thereof in one or moreaffected cells in a subject having a TDP-43 proteinopathy)) inassociation with the required pharmaceutical carrier and optionally inassociation with the other agent. Suitable administration frequenciesare described elsewhere herein.

Following administration of one or more of the compounds describedherein (or pharmaceutical compositions thereof) to a subject (e.g., ahuman patient), the efficacy of the treatment in ameliorating one ormore symptoms of a TDP-43 proteinopathy can be assessed by comparing thenumber and/or severity of one or more symptoms presented by a patientbefore and after treatment. Alternatively, where administration of thecompounds is used to prevent the occurrence of a TDP-43 proteinopathy,treatment efficacy can be assessed as a delay in presentation of, or afailure to present, one or more symptoms of a TDP-43 proteinopathy. Theefficacy of a treatment (e.g., a compound or composition describedherein) over time (e.g., a progressive improvement) in ameliorating oneor more symptoms of a TDP-43 proteinopathy can be determined byassessing, e.g., the number or severity of one or more symptoms atmultiple time points following treatment. For example, a subject (e.g.,a patient) can have an initial assessment of the severity of his or herdisorder (e.g., the number or severity of one or more symptoms of aTDP-43 proteinopathy), administered treatment, and then assessedsubsequently to the treatment two or more times (e.g., at one week andone month; at one month at two months; at two weeks, one month, and sixmonths; or six weeks, six months, and a year). Where one or morecompounds or compositions are administered to a subject for a limitedperiod of time (e.g., a predetermined duration) or number ofadministrations, the effect of treatment on ameliorating one or moresymptoms of a TDP-43 proteinopathy can be assessed at various timepoints after the final treatment. For example, following the lastadministration of a dose of one or more compounds, the number orseverity of a patient's symptoms can be assessed at 1 month (e.g., at 2months, at 6 months, at one year, at two years, at 5 years or more)subsequent to the final treatment.

The efficacy of a treatment with one or more compounds (or compositions)described herein on one or more symptoms of a TDP-43 proteinopathy canbe assessed as a monotherapy or as part of a multi-therapeutic regimen.For example, the compound(s) can be administered in conjunction withother clinically relevant treatments for TDP-43 proteinopathy including,but not limited to, physical or respiratory therapy, speech-languagetherapy, a decongestant, an anticonvulsant, and/or Rilutek®.

A compound or pharmaceutical composition thereof described herein can beadministered to a subject as a combination therapy with anothertreatment (another active ingredients), e.g., a treatment for a TDP-43proteinopathy such as ALS. For example, the combination therapy caninclude administering to the subject (e.g., a human patient) one or moreadditional agents that provide a therapeutic benefit to the subject whohas, or is at risk of developing, (or suspected of having) a TDP-43proteinopathy. Thus, the compound or pharmaceutical composition and theone or more additional agents are administered at the same time.Alternatively, the compound can be administered first in time and theone or more additional agents administered second in time. The one ormore additional agents can be administered first in time and thecompound administered second in time. The compound can replace oraugment a previously or currently administered therapy. For example,upon treating with a compound of the invention, administration of theone or more additional agents can cease or diminish, e.g., beadministered at lower levels. Administration of the previous therapy canalso be maintained. In some instances, a previous therapy can bemaintained until the level of the compound (e.g., the dosage orschedule) reaches a level sufficient to provide a therapeutic effect.The two therapies can be administered in combination.

It will be appreciated that in instances where a previous therapy isparticularly toxic (e.g., a treatment for a TDP-43 proteinopathy thatcarries significant side-effects) or poorly tolerated by a subject(e.g., a patient), administration of the compound can be used to offsetand/or lessen the amount of the previous therapy to a level sufficientto give the same or improved therapeutic benefit, but without thetoxicity.

In some instances, when the subject is administered a compound orpharmaceutical composition of the invention, the first therapy ishalted. The subject can be monitored for a first pre-selected result,e.g., an improvement in one or more symptoms of a TDP-43 proteinopathysuch as any of those described herein (e.g., see above). In some cases,where the first pre-selected result is observed, treatment with thecompound is decreased or halted. The subject can then be monitored for asecond pre-selected result after treatment with the compound is halted,e.g., a worsening of a symptom of a TDP-43 proteinopathy. When thesecond pre-selected result is observed, administration of the compoundto the subject can be reinstated or increased, or administration of thefirst therapy is reinstated, or the subject is administered both acompound and first therapy, or an increased amount of the compound andthe first therapeutic regimen.

In addition, while the invention is not limited by any particular theoryor mechanism of action, because the compounds identified herein canfunction at the molecular level to correct the TDP-43 proteinopathy,assessing the effect of a therapy on subject having a TDP-43proteinopathy can be done by assessing, e.g., whether a decrease in, orprevention of, the formation, deposition, accumulation, or persistenceof ubiquitinated, hyperphosphorylated forms of TDP-43 protein and/orC-terminal fragments thereof in one or more affected cells in a subjecthaving (or suspected of having a TDP-43 proteinopathy) occurred.

When the terms “prevent,” “preventing,” or “prevention” are used hereinin connection with a given treatment for a TDP-43 proteinopathy, theymean that the treated subject either does not develop a clinicallyobservable level of the TDP-43 proteinopathy at all (e.g., the subjectdoes not exhibit one or more symptoms of the TDP-43 proteinopathy or, atthe molecular level, does not develop aggregate TDP-43 or the formation,deposition, accumulation, or persistence of ubiquitinated,hyperphosphorylated forms of TDP-43 protein and/or C-terminalfragments), or the condition develops more slowly and/or to a lesserdegree (e.g., fewer symptoms or a lower amount of aggregate TDP-43) inthe subject than it would have absent the treatment. These terms are notlimited solely to a situation in which the subject experiences no aspectof the TDP-43 proteinopathy whatsoever. For example, a treatment will besaid to have “prevented” the TDP-43 proteinopathy if it is given to asubject at risk of developing a TDP-43 proteinopathy and results in thesubject's experiencing fewer and/or milder symptoms of the proteinopathythan otherwise expected. A treatment can “prevent” an TDP-43proteinopathy (e.g., ALS) when the subject displays only mild overtsymptoms of the TDP-43 proteinopathy. “Prevention” does not imply thatthere must have been no symptoms of a TDP-43 proteinopathy, or noaggregate TDP-43 or formation, deposition, accumulation, or persistenceof ubiquitinated, hyperphosphorylated forms of TDP-43 protein and/orC-terminal fragments in any cell of a subject.

The following are examples of the practice of the invention. They arenot to be construed as limiting the scope of the invention in any way.

EXAMPLES Yeast Models of TDP-43 Aggregation and Toxicity

Several yeast strains were generated that enable constitutive orinducible expression of full-length human TDP-43. To facilitate theanalysis of TDP-43 aggregation in vivo, GFP-tagged versions of humanTDP-43 were also made. GFP-TDP-43 was expressed in yeast using agalactose-inducible promoter. Expression of GFP-tagged TDP-43 resultedin the formation of multiple cytoplasmic inclusions in yeast cells (FIG.1). These findings demonstrate that yeast can be used as a model systemfor the formation of insoluble cytoplasmic TDP-43 aggregates.

The effect of TDP-43 accumulation on cellular toxicity was alsodetermined. Yeast cells were transformed with either an empty vector ora galactose-inducible TDP-43 expression plasmid. Serial dilutions oftransformants were spotted on glucose or galactose and growth wasassessed after two days. Expression of TDP-43 (in transformants grown ongalactose plates) was found to be highly toxic to yeast cells (FIG. 2).These findings demonstrate that yeast can be used as a model system forTDP-43-induced cellular toxicity.

OTHER EMBODIMENTS

It is to be understood that, while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention. Other aspects, advantages, and modifications of the inventionare within the scope of the claims set forth below.

What is claimed is:
 1. A method of inducing toxicity in a yeast cell,the method comprising: providing a yeast cell comprising an expressionconstruct comprising an inducible promoter operably linked to a nucleicacid encoding a polypeptide comprising a naturally occurring TARDNA-binding protein 43 (TDP-43) protein, wherein the naturally occurringTDP-43 protein comprises an amino acid sequence that is at least 95%identical to amino acid residues 252-414 of SEQ ID NO:1, whereinexpression of the nucleic acid and production of the polypeptide resultsin a decrease in growth or viability of the cell; and inducing a levelof expression of the nucleic acid in the yeast cell that is toxic to theyeast cell.
 2. A method of identifying a compound that prevents orsuppresses TDP-43-induced toxicity, the method comprising: culturing ayeast cell in the presence of a candidate agent and under conditionsthat allow for expression of the nucleic acid at a level that, in theabsence of the candidate agent, is sufficient to induce toxicity in theyeast cell, wherein the yeast cell comprises an expression constructcomprising a promoter operably linked to a nucleic acid encoding apolypeptide comprising a naturally occurring TAR DNA-binding protein 43(TDP-43) protein, wherein the naturally occurring TDP-43 proteincomprises an amino acid sequence that is at least 95% identical to aminoacid residues 252-414 of SEQ ID NO:1, wherein expression of the nucleicacid and production of the polypeptide results in a decrease in growthor viability of the cell; measuring cell growth or viability in thepresence of the candidate agent; and comparing cell growth or viabilitymeasured in the presence of the candidate agent to cell growth orviability in the absence of the candidate agent, wherein if cell growthor viability is increased in the presence of the candidate agent ascompared to in the absence of the candidate agent, then the candidateagent is identified as a compound that prevents or suppressesTDP-43-induced toxicity.
 3. A method of identifying a compound thatinhibits the formation or maintenance of cytoplasmic inclusions ofTDP-43, the method comprising: providing a yeast cell comprising anexpression construct comprising a nucleic acid encoding a polypeptidecomprising a naturally occurring TDP-43 protein, wherein the naturallyoccurring TDP-43 protein comprises an amino acid sequence that is atleast 95% identical to amino acid residues 252-414 of SEQ ID NO:1;culturing the yeast cell in the presence of a candidate agent and underconditions that allow for expression of the nucleic acid at a levelthat, in the absence of the candidate agent, is sufficient to result inthe formation or maintenance of cytoplasmic inclusions of the protein;measuring the formation or maintenance of cytoplasmic inclusions of theprotein in the presence of the candidate agent; and comparing theformation or maintenance of cytoplasmic inclusions of the proteinmeasured in the presence of the candidate agent to the formation ormaintenance of cytoplasmic inclusions of the protein in the absence ofthe candidate agent, wherein if formation or maintenance of cytoplasmicinclusions of the protein is decreased in the presence of the candidateagent as compared to in the absence of the candidate agent, then thecandidate agent is identified as a compound that inhibits the formationor maintenance of cytoplasmic inclusions of TDP-43.
 4. The method ofclaim 2, wherein expression of the nucleic acid and production of thepolypeptide renders the cell non-viable.
 5. The method of claim 2,wherein the expression construct is integrated in the genome of theyeast cell.
 6. The method of claim 2, wherein the expression constructis an integrative plasmid.
 7. The method of claim 6, wherein theintegrative plasmid is pRS303, pRS304, pRS305, or pRS306.
 8. The methodof claim 2, wherein the promoter is an inducible promoter.
 9. The methodof claim 8, wherein the inducible promoter is GAL1-10, GAL1, GALL, GALS,GPD, ADH, TEF, CYC1, MRP7, MET25, TET, VP16, or VP16-ER.
 10. The methodof claim 2, wherein the promoter is a constitutively active promoter.11. The method of claim 2, wherein the polypeptide comprises SEQ IDNO:1.
 12. The method of claim 2, wherein the yeast is Saccharomycescerevisiae, Saccharomyces uvae, Saccharomyces kluyveri,Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha,Pichia pastoris, Pichia methanolica, Pichia kluyveri, Yarrowialipolytica, Candida sp., Candida utilis, Candida cacaoi, Geotrichum sp.,or Geotrichum fermentans.
 13. The method of claim 2, wherein thepolypeptide is a fusion protein comprising a detectable protein.
 14. Themethod of claim 13, wherein the detectable protein is a fluorescentprotein, an enzyme, or an epitope.
 15. The method of claim 13, whereinthe detectable protein is a fluorescent protein selected from the groupconsisting of a red fluorescent protein, green fluorescent protein, bluefluorescent protein, yellow fluorescent protein, and cyan fluorescentprotein.
 16. The method of claim 2, wherein at least one gene thatencodes a protein involved in drug efflux or cell permeability isdisrupted.
 17. The method of claim 16, wherein the at least one gene isPDR1, PDR3, PDR5, SNQ2, or ERG6.
 18. The method of claim 1, wherein theinducible promoter is GAL1-10, GAL1, GALL, GALS, GPD, ADH, TEF, CYC1,MRP7, MET25, TET, VP16, or VP16-ER.
 19. The method of claim 1, whereinthe naturally occurring TDP-43 protein is at least 95% identical to SEQID NO:1.
 20. The method of claim 2, wherein the naturally occurringTDP-43 protein is at least 95% identical to SEQ ID NO:1.
 21. The methodof claim 3, wherein the naturally occurring TDP-43 protein is at least95% identical to SEQ ID NO:1.
 22. The method of claim 1, wherein thepolypeptide comprises SEQ ID NO:1.
 23. The method of claim 3, whereinthe polypeptide comprises SEQ ID NO:1.
 24. The method of claim 1,wherein the polypeptide comprises amino acid residues 252-414 of SEQ IDNO:1.
 25. The method of claim 2, wherein the polypeptide comprises aminoacid residues 252-414 of SEQ ID NO:1.
 26. The method of claim 3, whereinthe polypeptide comprises amino acid residues 252-414 of SEQ ID NO:1.